Fig. 7
From: Increased yields and biological potency of knob-into-hole-based soluble MHC class II molecules
Biological potency of NPs displaying KIH- vs. c-jun/c-fos-based pMHCII. a Native (left) and denaturing (right) SDS-PAGE for NPs coated with a representative KIH-based pMHCII molecule. PFM denotes the iron oxide NP. MW: molecular weight markers; 1: 2 μg of KIH-based BDC2.5mi/IAg7 monomers; 2: 2.2 μL of PFM coated with KIH-based BDC2.5mi/IAg7 monomers; 3: 1.1 μL of PFM coated with KIH-based BDC2.5mi/IAg7 monomers; 4: 2 μg of KIH-based BDC2.5mi/IAg7 monomers; 5: 2.2 μL of PFM coated with KIH-based BDC2.5mi/IAg7 monomers; 6: 1.1 μL of PFM coated with KIH-based BDC2.5mi/IAg7 monomers. Electrophoretic behavior of the KIH-based pMHCII-NP compound shown herein is representative of at least 10 different pMHCII-NP preparations made using KIH-based pMHCIIs. b Luciferase activity induced by NPs coated with c-jun/c-fos- (‘conv’) or KIH-based BDC2.5mi/IAg7 monomers (normalized to that induced by soluble anti-CD3ε mAb) on Jurkat cells co-expressing mouse CD4, a BDC2.5 mi/IAg7-specific TCR and an NFAT-driven luciferase reporter. Data correspond to mean ± SEM of triplicates. c Percentages of BDC2.5mi/IAg7 tetramer-positive CD4+ T-cells in blood, spleen, pancreatic lymph nodes (PLN), mesenteric lymph nodes (MLN) and bone marrow (BM) from NOD mice treated (twice a week for 5 weeks) with NPs coated with c-jun/c-fos-based (‘conv’) BDC2.5mi/IAg7 or KIH-based BDC2.5mi/IAg7 monomers (20 μg pMHC/dose). Data correspond to average ± SEM values from 4 mice/group. d Cytokine profile of the tetramer+ cells isolated from the mice in (c). Tetramer+ cells were challenged with anti-CD3/anti-CD28 mAb-coated beads for 3 days and the supernatants assayed for cytokine content using Luminex technology. Data correspond to average ± SEM values of cells isolated from 4 mice/group. P values were calculated via Mann–Whitney U and are considered significant if P < 0.05. e Luciferase activity induced by NPs coated with KIH-based BDC2.5 mi/IAg7 pMHCs carrying a mouse or a human Fc-based KIH (normalized to that induced by soluble anti-CD3ε mAb) on Jurkat cells co-expressing mouse CD4, a BDC2.5mi/IAg7-specific TCR and an NFAT-driven luciferase reporter. Data correspond to mean ± SEM of triplicates. Source data for panels (b–e) are provided as a Source Data File
